Composition for treating Duchenne muscular dystrophy based on metabolic adaptation

Abstract

The invention discloses a composition for treating Duchenne muscular dystrophy based on metabolic adaptation, and belongs to the technical field of biomedical engineering. The composition comprises two AAV vectors, wherein the first AAV vector comprises a mini utrophin gene, and the second AAV vector comprises an AMPK alpha 2 gene. According to the invention, a MyoAAV serotype is adopted as a carrier, and an MHCK7 promoter is utilized to realize specific high-efficiency expression of skeletal muscle. The two AAV viruses are jointly delivered into the body of a patient in an intravenous injection mode, the mini utrophin can partially recover the amyotrophin function, and the AMPK alpha 2 improves the muscle energy metabolism defect by adjusting a metabolic pathway. Experimental results in an mdx mouse model show that the double-target-point combined treatment scheme significantly improves the skeletal muscle pathological structure, reduces the serum CK level and improves the motor function to be close to the normal level. Safety evaluation of cynomolgus monkeys shows that the composition is good in safety. The limitation of existing single-target treatment is broken through, and a safer and more effective gene treatment strategy is provided for DMD patients.

Description

Technical Field

[0001] This invention relates to biomedical engineering, and more specifically to a composition for treating Duchenne muscular dystrophy based on metabolic adaptation. Background Technology

[0002] Duchenne muscular dystrophy (DMD) is an X-linked recessive inherited muscle disease caused by mutations in the DMD gene, resulting in the absence or dysfunction of dystrophin, leading to progressive muscle atrophy and weakness. Currently, there is no cure, but gene therapy offers new hope. Gene replacement therapy using adeno-associated virus (AAV) vectors is the most advanced and clinically applicable treatment strategy. The basic idea is to introduce a shortened but functional micro-dystrophin gene into the patient's body via an AAV vector, thereby partially restoring the function of dystrophin. Most current gene therapy strategies for DMD involve truncating and optimizing the delivery of dystrophin. A representative drug of this strategy is Elevidys, which received accelerated approval from the FDA in 2023 for specific DMD patients. However, the subsequent Phase III EMBARK trial failed to meet its primary endpoint and resulted in patient deaths, raising concerns about its safety and efficacy among the market and patients.

[0003] Dystrophin and Utrophin are structurally homologous and functionally highly similar proteins. In the pathological state of DMD, utrophin undergoes compensatory increases in the body, partially compensating for the muscle dysfunction caused by dystrophin deficiency; however, the body's own activation and compensation are insufficient to improve the disease. Previous published studies have confirmed that utrophin is a safe, effective, and low-immunogenic therapeutic target. Therefore, increasing the expression level of utrophin in patients through drugs or other means to achieve the goal of treating DMD is one feasible approach. However, based on extensive basic and clinical research data worldwide, simply increasing the expression of dystrophin protein in patients has not met the primary clinical endpoint, and the improvement in patients' clinical motor performance and lifespan remains unsatisfactory.

[0004] While the pathogenesis of DMD is caused by genetic defects, there is a certain correlation between the pathological manifestations of DMD and metabolism. Studies have shown that muscle cells in DMD patients have defects in energy metabolism, leading to impaired muscle function. These metabolic abnormalities may be related to mutations in the DMD gene encoding dystrophin. The absence or dysfunction of dystrophin affects metabolic pathways within muscle cells, including glycogen synthesis, fatty acid oxidation, cellular ion channels, and mitochondrial function. AMPK, as a key component in regulating metabolism, plays an important role in the progression of many diseases and the development of biological functions. Numerous studies have shown that AMPK also plays an important role in DMD and is a potential therapeutic target.

[0005] Currently, single-target therapy for DMD is the mainstream approach in clinical development. Its advantages include a clear mechanism, a well-defined research pathway, and easier in-depth scientific research targeting a specific target (such as delivery of miniature dystrophin or exon skipping therapy), leading to relatively direct pharmacodynamic and toxicological assessments. However, the limitations of single-target therapy are also evident. DMD is a complex, multi-systemic, progressive disease, and single interventions often only address part of the problem, failing to completely halt disease progression. Whether it's the already approved Elevidys or treatments in development and early clinical stages (such as the Eli Lilly PF-06939926 trial, which was terminated due to failure to meet the primary clinical endpoint), the limitations of single-target therapy are evident.

[0006] Therefore, there is an urgent need to develop new treatment strategies that target multiple key pathways and multiple pathological mechanisms of the disease through combination therapy, so as to maximize synergistic effects and efficacy, thereby more effectively improving the survival status and quality of life of DMD patients. Summary of the Invention

[0007] In view of the problems existing in the background technology, the present invention provides a composition for treating Duchenne muscular dystrophy based on metabolic adaptation, which is a gene therapy for DMD based on the dual-target combination therapy of modified mini utrophin and AMPK.

[0008] The technical approach adopted in this invention is applicable to all types of DMD patients, regardless of the specific type of gene mutation, because it does not rely on repairing the original mutated gene, but provides a highly functional alternative.

[0009] This invention is implemented through the following technical solutions: The first aspect of the present invention provides a composition for treating Duchenne muscular dystrophy based on metabolic adaptation, the composition comprising: The first AAV viral vector contains the mini utrophin gene; The second AAV viral vector contains the AMPKα2 gene.

[0010] In some embodiments, both the first AAV viral vector and the second AAV viral vector are MyoAAV serotypes.

[0011] In some implementations, both the first AAV virus vector and the second AAV virus vector contain the MHCK7 promoter.

[0012] The MyoAAV serotype was selected, and the MHCK7 promoter was used to achieve skeletal muscle-specific and efficient expression. A single viral vector was used to express mini utrophin and AMPKα2 separately, producing AAV viruses of acceptable quality.

[0013] In some embodiments, the nucleotide sequence of the mini utrophin gene is shown in SEQ ID NO.1; and the nucleotide sequence of the AMPKα2 gene is shown in SEQ ID NO.2.

[0014] In some embodiments, the dosage ratio of the first AAV viral vector to the second AAV viral vector is (1~5):1, preferably 2:1.

[0015] In some embodiments, the dosage of the first AAV viral vector is 1 × 10⁻⁶. 13 ~5×10 13 vg / kg, preferably 2×10 13 vg / kg; the dosage of the second AAV viral vector was 5 × 10 12 ~3×10 13 vg / kg, preferably 1×10 13 vg / kg.

[0016] A second aspect of the present invention discloses a pharmaceutical composition comprising the above-described composition and a pharmaceutically acceptable carrier or excipient.

[0017] In some embodiments, the pharmaceutically acceptable carrier or excipient includes, but is not limited to, buffer solutions, stabilizers, isotonic modifiers, preservatives, etc.

[0018] In some implementations, the application is applicable to all types of DMD patients, regardless of the specific type of gene mutation.

[0019] The third aspect of this invention discloses the use of the above-described composition or the above-described pharmaceutical composition in the preparation of a medicament for treating Duchenne muscular dystrophy.

[0020] A fourth aspect of the present invention discloses a method for treating Duchenne muscular dystrophy, the method comprising administering to a patient a therapeutically effective amount of the above-described composition or the above-described pharmaceutical composition.

[0021] In some embodiments, the administration method is intravenous injection; the patient is a DMD patient of any gene mutation type.

[0022] The beneficial effects of this invention are: 1. This invention is the first to propose a dual-target combined therapy strategy based on miniutrophin and AMPKα2 for the gene therapy of DMD. This strategy differs from existing single-target treatments, simultaneously addressing two key pathological aspects: the root cause of the disease (deficiency of dystrophin) and metabolic disorder, achieving a comprehensive therapeutic goal. Miniutrophin compensates for the loss of dystrophin function, restoring muscle membrane stability and the structural integrity of muscle cells; AMPKα2 regulates energy metabolism, improving metabolic abnormalities in muscle cells and reducing oxidative stress and chronic inflammation. The combined use of these two drugs produces a synergistic effect, more effectively blocking multiple stages of the DMD pathological cascade, thereby better slowing or halting disease progression. This invention also innovatively uses the novel MyoAAV serotype as a delivery vector, demonstrating extremely high application potential in terms of safety and efficacy.

[0023] 2. Experimental results in the MDX mouse model showed that this dual-target combined treatment regimen significantly improved skeletal muscle pathological structure, reduced serum CK levels, and enhanced motor function to near-normal levels. Safety evaluation in cynomolgus monkeys showed that the composition had good safety. This invention overcomes the limitations of existing single-target therapies, providing a safer and more effective gene therapy strategy for DMD patients.

[0024] 3. The treatment strategy of this invention does not rely on repairing the patient's original mutated gene, but rather works by providing functional replacement proteins and improving the metabolic environment. Therefore, it is applicable to all types of DMD patients, regardless of the specific site and type of their DMD gene mutation. This feature significantly expands the potential treatment population and has a clear advantage over strategies such as exon skipping therapy that only targets specific mutation types. Attached Figure Description

[0025] The accompanying drawings are provided to further explain the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1The results show the detection levels of related proteins in different skeletal muscles; among them Figure 1 A represents the quadriceps femoris muscle. Figure 1 B represents the anterior cervical muscle. Figure 1 C represents the triceps brachii. Figure 1 D represents the detection results of related protein expression in the diaphragm; Figure 2 HE staining results of skeletal muscle from mice in different treatment groups; Figure 3 Serum CK expression levels in mice of different treatment groups; Figure 4 The results of behavioral tests on mice in different treatment groups; among them Figure 4 A represents the results of the relative gripping force test of the mouse's four limbs. Figure 4 B represents the results of the treadmill exhaustion test for mice. Figure 4 C represents the total time taken in the mouse pole-climbing experiment. Figure 4 D represents the turning time test result of the mouse pole climbing experiment. Figure 4 E represents the number of failed attempts in the mouse pole climbing experiment; Figure 5 The results of blood biochemical index testing in the safety evaluation of cynomolgus monkeys. Detailed Implementation

[0026] The technical solution of the present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0027] Example 1: Construction of AAV vector.

[0028] 1. Target selection and design.

[0029] We designed truncated utrophin and AMPKα2 genes with optimized sequences and optimized gene sequences to meet the packaging capacity of AAV vectors.

[0030] The optimized nucleotide sequence of the mini utrophin gene is shown in SEQ ID NO.1.

[0031] SEQ ID NO.1:

[0032] The optimized nucleotide sequence of the AMPKα2 gene is shown in SEQ ID NO.2.

[0033] SEQ ID NO.2:

[0034] 2. Vector construction and AAV virus production and purification.

[0035] The MyoAAV serotype was selected, and the MHCK7 promoter was used to achieve skeletal muscle-specific and efficient expression. A single viral vector was used to express mini utrophin and AMPKα2 separately, producing AAV viruses of acceptable quality.

[0036] The core process is divided into two main stages: upstream cell transfection and virus amplification, and downstream purification and stock solution preparation, as detailed below: I. Upstream: Triple plasmid transfection of 293 cells and viral amplification: Using the optimized gene core sequence described above, a professional manufacturing company was commissioned to construct and synthesize clearly traceable GOI and Rep-Cap plasmids. Using 293 cells in suspension culture as the host, AAV virus packaging and amplification were achieved through co-transfection with the three plasmids, providing virus harvesting solution for downstream applications. Key steps are as follows: Cell preparation: 293 cells were cultured in suspension in a 200L bioreactor, with the culture environment controlled (temperature 18-26℃, humidity ≤65%, referring to downstream environmental control logic) to ensure the cells reached the logarithmic growth phase (suitable density and viability to ensure transfection efficiency). Plasmid preparation: Three core plasmids required for AAV production were prepared—Rep-Cap plasmid (providing genes for viral replication and capsid protein synthesis), GOI plasmid (a recombinant vector carrying the target gene), and helper plasmid (providing auxiliary factors required for viral replication), ensuring that the plasmid purity and concentration met transfection requirements. Co-transfection procedure: Three plasmids were co-transfected into 293 suspension cells in a specific ratio using a transfection reagent. After mixing, suitable culture conditions (such as temperature, pH, dissolved oxygen, etc.) were maintained in the reactor to allow the plasmids to enter the cells and initiate virus packaging.

[0037] Virus amplification and harvesting: After transfection, the cells are cultured until the viral titer reaches its peak (usually 48-72 hours after transfection). The mixture of cells and supernatant is collected by centrifugation or filtration, which is the "AAV9 type AAV virus harvest solution produced by suspension in a 200L reactor", which serves as the starting material for downstream purification.

[0038] II. Downstream: Virus purification and stock solution preparation: Starting with the upstream harvested liquid, a purification process was performed to prepare AAV virus stock solution that met quality standards. The entire process was carried out under GMP conditions (Grade C basic, with Grade C + Grade A ventilation for critical steps), as detailed below: Clarification and filtration: Use a 0.04-20μm depth filter to filter the harvest liquid at a pump speed of 100-600rpm. First, rinse the filter with water for injection (≥552L), then rinse with RA solution until the pH difference between the outflow and the RA solution is ≤±0.5. After filtration, top-wash the pipeline / filter with RA solution until there is no red color. Collect the clarified liquid, control the pressure at the front end of the filter to ≤1.2bar, and take a sample for retention.

[0039] Ultrafiltration concentration: A 300 kDa ultrafiltration membrane pack was used. First, an integrity test was performed (gas flow rate ≤ 500 mL / min was acceptable). After the system was circulated with 0.5 M sodium hydroxide for 30 min and cleaned with CIP, it was equilibrated with RA solution (pH difference ≤ 0.5). The clarified liquid was concentrated 10-20 times at a flow rate of 2-8 L / min / m² and TMP ≤ 1.0 bar. Then, the filter was washed 5-7 times with an equal volume of RA solution. The concentrated liquid was collected, and the system was stored with 0.1 M sodium hydroxide.

[0040] Affinity chromatography: The column was clarified twice through a 0.45 μm filter (RA wash pH difference ≤ ±0.2, pressure ≤ 1.2 bar); A 400 mL column volume AAVX affinity chromatography column was used. After washing with 0.5 M sodium hydroxide for 20 min + CIP cleaning, the system was equilibrated with 0.1 M phosphoric acid (5 CV), RB solution (1 CV), and RA solution (5 CV) until the pH difference was ≤ 0.2 and the A260 baseline was stable; the sample was loaded at a flow rate of 0.2-1.0 CV / min, washed with RA solution for 5 CV until the A260 was stable, and then eluted with 100% RB solution. Collection began when the A260 reached 20 mAU and stopped when it dropped to 20 mAU. The collected solution was temporarily stored at 2-8℃, and the column was stored in 20% ethanol.

[0041] Ultracentrifugation: Add sodium chloride (final concentration +1M) and polysorbate 20 (final concentration 3%) to the affinity chromatography collection solution, dispense 30 mL / tube (make up the difference with RH solution if less than 30 mL); centrifuge at 49000 rpm and 18℃ for 90 min, and collect the bottom 3.5 mL of sample from each tube.

[0042] Anion chromatography: A 400 mL 50HQ anion exchange chromatography column was used. After washing with 0.5 M sodium hydroxide for 5 CV + CIP cleaning, the ultracentrifuged sample was diluted with 0.1 × RC solution to a conductivity ≤ 1.5 mS / cm. The column was equilibrated with RF solution (5 CV) → RD solution (1 CV) → RC solution (5 CV) until the pH difference was ≤ 0.2 and the conductivity was < 2 mS / cm. The sample was loaded at a flow rate of 0.2-1.0 CV / min. After washing with RC solution for 5 CV until the A260 was stable, a linear gradient elution of 0-100% RD solution was used for 20 CV. Collection began when the conductivity was > 2 mS / cm and the A260 reached 20 mAU, and stopped when it dropped to 20 mAU. The collected solution was temporarily stored at 2-8℃, and the column was stored in 20% ethanol.

[0043] Ultrafiltration replacement: A 100 kDa hollow fiber membrane column was used. First, an integrity test was performed (diffusion flow ≤ 2 mL / min / 0.1 m² is acceptable). After the system was circulated with 0.5 M sodium hydroxide for 30 min and cleaned with CIP, it was equilibrated with R012 buffer (pH 7.4 ± 0.5). The sample was concentrated to the target volume at a flow rate of 2-8 L / min / m² and TMP 2-5 PSI. Then, it was replaced 5-10 times with an equal volume of R012 buffer (permeate end pH 7.4 ± 0.3, conductivity ≤ ± 1.5 mS / cm). The replacement solution was collected, and the system was stored with 0.1 M sodium hydroxide.

[0044] Sterilization filtration and dispensing: Sterilize the 0.45 ± 0.2 μm filter and 25# silicone tubing, irradiate with UV light in a biosafety cabinet for 30 min + self-clean for 10 min, rinse the filter with R012 buffer (≥20 mL); filter the sample at a pump rate of 20-80 rpm, rinse the filter with R012 buffer (20-40 mL), and collect the stock solution; perform an integrity test on the filter (bubble point 3200-7000 mbar is acceptable), dispense the stock solution into PETG sterile bottles, and store below -60℃.

[0045] III. Quality Inspection of Finished Virus Products: 1. Appearance and physicochemical properties: Appearance: Visual inspection was used, based on the pharmacopoeia general chapter 0904. The expected acceptance standard is a colorless and transparent liquid, which is used to visually determine whether the original solution has any physical property problems such as abnormal color or turbidity.

[0046] pH: Detected by potentiometric method, according to Pharmacopoeia General Chapter 0631 and USP. <791> The specific acceptance criteria are determined based on the formulation requirements to ensure that the pH of the stock solution meets the requirements of subsequent formulations and the usage environment.

[0047] Osmoregulation concentration: using the freezing point method, according to Pharmacopoeia General Chapter 0632 and USP <785> Acceptance standards vary depending on the formulation, ensuring that the osmotic pressure of the original solution is compatible with the biological environment, thus avoiding any impact on viral activity and safety of use.

[0048] 2. Virus characteristics and activity: Genome titer: The ddPCR method was used to quantify the number of AAV viral particles carrying the target gene in the stock solution, reflecting the viral load level.

[0049] Capsid titer: Two detection methods are used: ELISA for wild-type AAV and HPLC for modified AAV. Both are based on the company's internal control standards and the results must be reported. This is used to count the total amount of AAV capsid particles in the stock solution and to help evaluate the virus packaging efficiency.

[0050] Capsid protein identification and purity: Using the SDS-PAGE method, in accordance with the company's internal control standards, the capsid proteins VP1, VP2, and VP3 are required to show clear bands at their corresponding molecular weight positions, and the purity is ≥95%, in order to identify the target capsid protein and control the content of impurity proteins.

[0051] Empty shell ratio: Using the AUC (analytical ultracentrifugation) method, based on the company's internal control standards, the expected empty shell ratio is ≤10%, which is used to control the proportion of empty capsid particles without target genes and ensure the effective infectivity of the virus.

[0052] AAV purity (HPLC): Using the HPLC method, in accordance with the company's internal control standards, the AAV monomer content is required to be ≥90% to reduce the impact of viral aggregates on purity and activity.

[0053] Virus aggregation: Using SEC-HPLC (size exclusion high performance liquid chromatography) method, in accordance with the company's internal control standards, the expected aggregation ratio is <10%, ensuring that virus particles exist in monomer form and avoiding the decrease in activity or increase in immunogenicity caused by aggregation.

[0054] 3. Safety-related residues: Host cell DNA residue: Using qPCR, in accordance with the company's internal control standards, the test results must be reported to control the amount of residual host cell genomic DNA and reduce the potential risk of insertion mutations.

[0055] Host cell E1A residue: Based on the company's internal control standards and measured by ddPCR, the residue level is required to be <10. 7 copies / 10 12 vg, E1A gene is related to cell transformation, and its residues must be strictly controlled to ensure safe use.

[0056] Host cell protein residues: Using ELISA, based on the company's internal control standards, the expected residue level is <50 ng / 10. 13 vg reduces the immune response triggered by host cell proteins.

[0057] Plasmid DNA residue: Using ddPCR, in accordance with the company's internal control standards, the results must be reported to control the residue of free plasmid DNA that was not packaged into the virus during the production process and to avoid the impact of irrelevant nucleic acids on the body.

[0058] Nuclease residue: According to the ELISA method and the General Pharmacopoeia 0512, the residue level should be <10 ng / 10 13 vg prevents nucleases from degrading the viral genome and ensures the stability of the viral structure.

[0059] Affinity ligand residue: Using HPLC, based on the company's internal control standards, the expected residue level is <100 ng / 10 13VG controls the residual ligands of affinity chromatography packing material during downstream purification, reducing the risk of impurity introduction.

[0060] Transfection reagent residue: Using HPLC and in accordance with the company's internal control standards, the residue level is required to be <10ppm to reduce the cytotoxicity or immunogenicity of transfection reagents (such as PEI).

[0061] Tween 20 residue: Using HPLC and in accordance with the company's internal control standards, the expected residue level is <10ppm. This controls the residue of surfactants used in production to avoid adverse effects on viral activity or the body.

[0062] 4. Other critical security categories: Bacterial endotoxins: Utilized via gelation method, in accordance with General Pharmacopoeia 1143 and USP. <85> For intravenous injection scenarios, the endotoxin content is required to be <5 EU / (kg·h) (less than 1 hour is calculated as 1 hour) to prevent adverse reactions such as fever and shock caused by endotoxin.

[0063] Microbial limits (optional): Use microbial count methods, according to General Pharmacopoeia 1105 and USP. <61> The expected microbial count is ≤10 CFU / mL, controlling microbial contamination in the stock solution and ensuring safe use.

[0064] rcAAV (replicated intact AAV): Using CellAssay (cell culture method) combined with qPCR, and in accordance with the company's internal control standards, the rcAAV content must be <1 / 10-1. 8 vg and rcAAV have the ability to replicate, and their residues must be strictly controlled to prevent the virus from replicating uncontrollably in the body.

[0065] Example 2: Results of efficacy evaluation based on MDX mouse experiments.

[0066] Group design: MDX mice were injected with a combination of MyoAAV-MHCK7-mini utrophin and MyoAAV-MHCK7-AMPKα2 virus, with the MyoAAV-MHCK7-mini utrophin virus administered at a dose of 2 × 10⁻⁶. 13 The injection dose of MyoAAV-MHCK7-AMPKα virus is 1×10⁻⁶ vg / kg. 13 Male MDX mice aged 3-4 weeks were injected via tail vein at a dose of vg / kg as the MDX+U+A group (MDX+mini utrophin+AMPKα2); MyoAAV-MHCK7-mini utrophin tool virus was used at a dose of 2×10 13The injection dose was vg / kg. MDX mice aged 3-4 weeks were injected via tail vein as the mdx+utrophin injection group. MDX mice were injected with MyoAAV-MHCK7-AMPKα virus as the mdx+AMPKα group. MDX mice were used as the control group. All groups were housed in a standard SPF environment. Behavioral tests such as running, gripping, and pole climbing were started at the end of the 5th week (35 days) after the initial injection. The experiment ended at the end of the 6th week (42 days), and the mice were sacrificed for testing.

[0067] 1. Effects of combined treatment on the expression of skeletal muscle-related proteins in MDX mice.

[0068] Proteins were extracted from samples after standard freezing treatment, and the expression of miniutrophin, AMPKα, p-AMPKα, and DGC complex-related proteins was detected by Western blotting. The results showed that exogenous miniutrophin was successfully expressed in different skeletal muscles in both single and combined injection groups of miniutrophin. Compared with the mdx+miniutrophin group, the expression of AMPKα (~60kDa) and p-AMPKα (~60kDa) proteins in the quadriceps femoris, anterior cervical vertebrae, triceps brachii, and diaphragm of mice in the mdx+AMPKα and mdx+U+A groups was significantly increased (P≤0.05). Figure 1 Western blotting was used to detect the expression of αSG (~50 kDa) and γSG (~35 kDa) in the DGC complex. The results showed that, compared with the mdx+mini utrophin group, the expression of αSG and γSG in the mdx+AMPKα group and the mdx+U+A group was significantly increased in the quadriceps femoris muscle (P≤0.05). Figure 1 A); Compared with the mdx+AMPKα group, there was no significant difference in the expression of AMPKα and p-AMPKα proteins in the mdx+U+A group ( Figure 1 The results showed that after 6 weeks of combined delivery of exogenous mini utrophin and AMPKα to MDX mice via AAV virus, mini utrophin was successfully expressed in the skeletal muscle of MDX mice, AMPKα was successfully overexpressed, and p-AMPKα was activated. There was essentially no difference in the expression of αSG and γSG proteins in the skeletal muscle of different treatment groups.

[0069] 2. Effects of combined treatment on the histopathology of skeletal muscle tissue in MDX mice.

[0070] MDX mice aged 2 to 3-4 weeks were jointly delivered with mini utrophin and AMPKα. Six weeks later, tissue samples were prepared for HE staining. HE staining results showed that, compared with the MDX group, the typical DMD pathological features of skeletal muscle central nuclei, inflammatory infiltration, and skeletal muscle cell diameter variations disappeared in the MDX+U+A group. Figure 2 The results suggest that combined delivery of mini utrophin and AMPKα2 can significantly improve the pathological structure of skeletal muscle in MDX mice.

[0071] 3. Effect of combined treatment on serum CK in MDX mice.

[0072] MDX mice aged 2 to 3-4 weeks were jointly delivered with mini utrophin and AMPKα. Serum samples were collected at the end of the experiment, and serum CK levels were measured using a CK assay kit. Results showed that there was no significant difference in serum CK levels between the mdx+U+A group and the C57 group. Figure 3 The results indicate that the combined treatment significantly improved serum CK levels in MDX mice.

[0073] 4. Effects of combined treatment regimens on the behavior of MDX mice.

[0074] MDX mice aged 2-3-4 weeks were jointly administered mini utrophin and AMPKα and housed in a standard environment after injection. Behavioral tests, including grip strength testing, a one-time exhaustion treadmill test, and a pole climbing test, were conducted at the end of week 5 (35 days). The results showed no statistically significant differences in grip strength, one-time exhaustion treadmill test, and pole climbing ability between the mdx+U+A group and the C57 group. Figure 4 Compared with the mdx+mini utrophin group, the mdx+U+A group mice had a significantly longer single-run exhaustion time (P≤0.05). Figure 4 The mouse pole climbing experiment showed that there were no statistically significant differences among the C57 group, the mdx+mini utrophin group, the mdx+AMPKα2 group, and the mdx+U+A group. Figure 4 (CE), behavioral experiments suggest that combined treatment with mini utrophin and AMPKα2 significantly improves the motor function of mdx mice.

[0075] Example 3: Experimental results of AAV drug safety evaluation based on cynomolgus monkey animal model.

[0076] High and low doses of virus were delivered to normal cynomolgus monkey models, with the low-dose group receiving 6 × 10⁶ doses. 13 vg / kg, high-dose group was 9×10 13Vg / kg; Blood biochemical parameters of cynomolgus monkeys were measured at different time points after drug administration. The results showed that, compared with the normal control group, there were no differences in the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), creatine kinase (CK), total bilirubin (TBIL), urea (UREA), creatinine (CR), glucose (GLU), total cholesterol (TCH), triglycerides (TG), total protein (TP), albumin (ALB), globulin (GLOB), albumin / globulin ratio (A / G), Cl, Na, and K in the blood of the high- and low-dose groups. Figure 5 Relevant blood test results showed that the AAV drug had no effect on blood immune, metabolic and other related indicators in cynomolgus monkeys.

[0077] In summary, the experimental results demonstrate that the technical solution of this invention, which delivers miniutrophin and AMPKα2 genes via MyoAAV virus as a vector, has fully validated the efficacy of our drug treatment in a DMD model mouse (mdx mouse). Preclinical toxicology evaluation experiments have also verified the safety of the drug. This significantly addresses the current challenges of gene therapy in DMD.

[0078] Finally, it should be noted that the above embodiments are merely illustrative of several implementations of the present invention and are not intended to limit the scope of the invention. For those skilled in the art, any modifications, equivalent substitutions, or improvements made without departing from the concept of the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A composition for treating Duchenne muscular dystrophy based on metabolic adaptation, characterized in that, The composition comprises: The first AAV viral vector contains the mini utrophin gene; The second AAV viral vector contains the AMPKα2 gene.

2. The composition according to claim 1, characterized in that, Both the first AAV viral vector and the second AAV viral vector are MyoAAV serotype.

3. The composition according to claim 1, characterized in that, Both the first AAV virus vector and the second AAV virus vector contain the MHCK7 promoter.

4. The composition according to claim 1, characterized in that, The nucleotide sequence of the mini utrophin gene is shown in SEQ ID NO.1; the nucleotide sequence of the AMPKα2 gene is shown in SEQ ID NO.

2.

5. The composition according to claim 1, characterized in that, The dosage ratio of the first AAV viral vector to the second AAV viral vector is (1~5):

1.

6. The composition according to claim 1, characterized in that, The dosage of the first AAV viral vector was 1×10⁻⁶. 13 ~5×10 13 vg / kg; the dosage of the second AAV viral vector was 5 × 10 12 ~3×10 13 vg / kg.

7. A pharmaceutical composition, characterized in that, Includes the composition according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier.

8. The use of the composition according to any one of claims 1 to 6 or the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating Duchenne muscular dystrophy.

9. A method for treating Duchenne muscular dystrophy, characterized in that, The method includes administering to a patient a therapeutically effective amount of the composition according to any one of claims 1 to 6 or the pharmaceutical composition according to claim 7.

10. The method according to claim 9, characterized in that, The administration method is intravenous injection; the patient is a DMD patient of any gene mutation type.

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